Primary immunodeficiency diseases (PIDDs) are a large group of genetic disorders of the immune system. These disorders vary in the severity and spectrum of symptoms, but without effective and early treatment, they can be fatal. Currently, there is no reliable screening assay for early diagnosis of PIDDs. The goal of our proposal is to develop and validate a specific and quantitative assay that will simultaneously identify multiple PIDDs using a small volume of blood. We previously developed a novel proteomic screening method using Selected Reaction Monitoring-Mass Spectrometry (SRM-MS) to simultaneously identify low-abundant specific signature peptides derived from the transmembrane protein cluster of differentiation 3 (CD3?) and the intracellular proteins, Wiskott-Aldrich syndrome protein (WASP) and Bruton's tyrosine kinase (BTK) as markers of three life-threatening PIDDs; severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), and X-linked Agammaglobulinemia (XLA). The objective of this application is to improve the sensitivity of our novel approach by developing peptide immunoaffinity enrichment coupled to selected reaction monitoring- mass spectrometry (immuno-SRM-MS) to quantify a panel of biomarkers in infant blood to facilitate the early detection and diagnosis of multiple life-threatening PIDDs and validate these panels of biomarkers for clinical implementation. Our Aims are to: 1. Expand the existing panel of screenable PIDDs by identifying proteotypic signature peptides for 9 additional conditions using SRM-MS. These PIDDs include ADA- deficient SCID, MHC class II deficient SCID, Hyper IgE recurrent infection syndrome, 2 Common Variable Immunodeficiency Disorders (CVIDs) 3 and 8 , Ataxia Telangiectasia, Hemophagocytic lymphohistiocytosis, X-linked lymphoproliferative disease, and X-linked chronic granulomatous disease. We will use human cell lines to select signature peptides for these PIDDs and fully optimize SRM-MS conditions. 2. Increase sensitivity of the SRM-MS assay for PIDDs by coupling it with peptide immunoaffinity enrichment. We will employ immuno-SRM procedures for measurements of signature peptides for the 15 target proteins for various PIDDs to improve the sensitivity of our assay for clinical implementation. We will measure performance metrics for each assay by generating a response curve. 3. Evaluate the ability of a multiplex immuno-SRM approach to correctly identify patients with specific immunodeficiencies in a larger set of clinical samples. Our multiplex immuno-SRM assay will be deployed on human clinical samples retrospectively and prospectively collected by the Seattle Children's Immunology Diagnostic Laboratory.